氯代亚甲基硫色满酮抗肿瘤作用机制研究

发布时间：2018-08-01 13:52

【摘要】：本论文对氯代亚甲基硫色满酮抗肿瘤机制进行了初步研究。以人肝癌HepG-2细胞，人宫颈癌Hela细胞为模型，采用台盼蓝染色法检测CMCT、CMMT对细胞增殖的影响；Hoechst33258染色，流式细胞术(FCM)检测CMCT、CMMT对细胞凋亡的影响；ELISA法检测CMMT对凋亡相关蛋白表达的影响；RT-PCR方法检测凋亡相关基因转录水平的变化；ELISA法检测CMMT对小鼠巨噬细胞分泌因子激活作用。实验结果表明CMCT、CMMT能够明显抑制HepG-2和Hela细胞的增殖，表现为低浓度抑制，高浓度致死；Hoechst33258荧光染色和流式细胞术检测结果显示CMCT、CMMT可诱导肿瘤细胞凋亡；CMMT作用24h后，caspase-3，-8和-9的蛋白含量增高，并呈浓度依赖性；ELISA法检测显示CMMT可使HepG-2细胞中死亡受体TNFR1、Hela细胞中DR3表达量显著增高；CMMT可以上调促调往基因Bax、p53的表达，下调抑凋亡基因Bcl-2的表达。基于硫色满酮类化合物可能具有免疫促进的猜测，我们对CMMT可否激活巨噬细胞进行了实验。实验结果表明，CMMT可激活小鼠的原代腹腔巨噬细胞，提高TNF-α、IL-6、IL-12的分泌量，具有类似LPS的作用，提示其有可能是TLRs的激动剂。以上结果表明CMMT的抗癌机制可能是通过死亡受体和线粒体两个途径诱导肿瘤细胞凋亡；并能通过免疫激活作用激活体内先天免疫系统，，间接发挥抗肿瘤作用。
[Abstract]:In this paper, the anti-tumor mechanism of chloromethylene thiolone was studied. The effect of HepG-2 on cell proliferation was detected by trypan blue staining, and the apoptosis was detected by flow cytometry (FCM) (FCM). Effects of CMMT on the expression of apoptosis-related proteins by ELISA; RT-PCR method was used to detect the transcription level of apoptosis-related genes. Elisa was used to detect the activation of macrophage secreting factors by CMMT. The results showed that the proliferation of HepG-2 and Hela cells was significantly inhibited by CMCTT. The results of high concentration lethal Hoechst33258 fluorescence staining and flow cytometry showed that CMCTMT-CMMT could induce apoptosis of tumor cells. The protein levels of caspase-3, caspase-8 and -9 were increased 24 hours after treatment with CMMT. The results of concentration dependent Elisa showed that CMMT could significantly increase the expression of DR3 in HepG-2 cell line TNFR1 and Hela cells. CMMT could up-regulate the expression of Bax-p53 gene. The expression of Bcl-2 was down-regulated. Based on the hypothesis that thiolemone compounds may be immune-promoting, we have studied whether CMMT can activate macrophages. The results showed that CMMT could activate the primary peritoneal macrophages of mice and increase the secretion of TNF- 伪 and IL-6 IL-12, which was similar to that of LPS, suggesting that it might be an agonist of TLRs. These results suggest that the anticancer mechanism of CMMT may be induced apoptosis of tumor cells by death receptor and mitochondria, and can activate innate immune system through immune activation, and indirectly play an anti-tumor role.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

【参考文献】